SUMMARY Vascular grafts that are currently used in small-diameter arterial bypass or in AV access for dialysis are not ideal and have significant failure rates. Tissue-engineered vascular grafts (TEVGs) using autologous mesenchymal stem cells (MSCs) show promise, but have two main limitations that may prevent their clinical translation. First, patients at high risk for cardiovascular disease - such as the elderly and diabetics - have dysfunctional MSCs which may not be able to yield a viable TEVG. Second, the use of any cell type that requires extended culture expansion ? including MSCs ? opens the door to the risk of cellular contamination or transformation, as well as high costs and a substantial waiting time before a TEVG can be fabricated. The overall goal of the proposed work is to develop a novel, clinically-viable, rapidly-fabricated, cell-free TEVG. The overarching hypothesis of this proposal is that secreted factors from human MSCs can be harvested, packaged, and delivered by ?artificial MSCs? (artMSCs) that can then replace the paracrine activity of the MSCs in our TEVG. Three specific aims will address our hypotheses: Aim 1: Develop ?artificial stem cells? comprised of families of degradable and tunable microspheres loaded with conditioned media from human MSCs. The goal of the artMSCs is to replace the need for cells in our TEVG, but maintain their critical secreted factors. We expect that we can encapsulate and time-release the soluble factors from biologically active human MSC using PLGA microspheres. We will validate this by using the released factors to drive vascular cell migration and proliferation. Aim 2: Tune the artMSCs in order to most optimally replace the time course of MSC secretory activity and residency within an implanted TEVG. This aim will have two parts: A) An in-vivo time course evaluation of MSC-based TEVGs in a rat model, monitoring presence of implanted MSCs and timing of host SMC and endothelial cell recruitment and remodeling. B) Utilize this timing information to fabricate discrete families of artMSCs that will each release their cargo at different times. We expect the net effect of our artMSC families to approximate the paracrine activity of actively secreting MSC present within the remodeling TEVG. Aim 3: Test the in vivo efficacy of a TEVG comprised of our artMSCs. For this aim, we will seed the families of artMSCs into biodegradable scaffolds and evaluate them as a TEVG in a rat model. We expect that a microsphere-loaded, acellular scaffold will be at least as effective as a TEVG as a scaffold loaded with MSCs. The TEVGs will be assessed by metrics of success including patency and an artery-like composition. An innovative therapy based on secreted factors from standardized human MSC cell lines (i.e., from healthy patients) would offer a uniform treatment strategy from patient to patient than an inherently variable autologous cell-based strategy. The cell-free nature of our approach is more easily translatable to the clinic, and the cost and time spent harvesting cells from individual patients would be eliminated.